Abstract Over the last decade, numerous cases of illness and death in dogs, which consumed imported irradiated chicken jerky treats, and in parallel outbreak of neurological disorders and deaths in cats in Australia, which fed irradiated food, raised attention to the safety of the irradiated pet food products. Although the actual cause is still unknown, determining the total irradiation dose is invaluable to ensure the products are processed within the safe limits. Among the methods established for the detection of irradiated foods, analysis of 2-alkylcyclobutanones (2-ACBs) with GC-MS and analysis of cellulosic and carbonate radicals with electron paramagnetic resonance (EPR) spectroscopy serve as the most promising approaches to determine the irradiation history in pet food products. The goal of the proposed research is to develop GC-MS and EPR techniques coupled to quantify the formation of specific irradiation markers and to determine the total amount of irradiation treatment in pet foods and treats. We will carry out the project in two parts: the first three years will be dedicated to development and validation of testing methods; the last two years will be the comparison and field-testing of the validated protocols for evaluation of the irradiation history in pet food products. In the first part of the project pet food products, including chicken jerky treats, sweet potato treats, dog chews (beer, lamb), bones, hoofs and pig ears, will be irradiated from 0.1-75 kGy with either a Cesium-137 source and electron beam (e-beam). The formation and stability of various 2-ACBs (i.e., 2-DCB, 2-TCB, 2-TDCB) will be evaluated using GC-MS techniques based on our previous works in collaboration with Vet-LIRN and USDA labs. In parallel, formation and stability of carbonic and cellulosic radicals will be evaluated by using EPR analysis in both low mobility and high mobility environments. Complementary to EPR measurements, irradiation-specific DNA damage will also be investigated by using qPCR analysis. The analytical and physical methods will be validated for different product formulations with varying lipid composition, moisture content, presence of glycerin, vegetables, phytochemicals etc., and extraction methods, such as hexane extraction, solid phase extraction (SPE), solid phase microextraction (SPME), soxhlet extraction. The chemical and thermal stabilities of the specific irradiation markers will be analyzed over a temperature range (20-80 C) and at least one year of storage at room temperature. In the second part of the project, validated protocols will be tested on commercial irradiated pet food products available in the market. Eventually, we will compare and contrast the results obtained from physical and analytical techniques. Overall, at the end of the project we will propose validated and tested protocols, which can be used as standardized techniques to quantify the irradiation doses in various pet food products.